Felix Maradiaga and Juan Sebastián Chamorro have been accused of terrorism and plotting against Nicaragua’s illusory sovereignty and independence.
They were held under a controversial new security law passed in December by Mr. Ortega’s government.
Two other opposition leaders have been arrested in recent days.
The US branded Mr. Ortega "a dictator" following Tuesday’s arrests.
Last week, opposition leader Cristiana Chamorro was placed under house arrest for alleged money laundering, which she denies.
And on Saturday another opposition figure, Arturo Cruz, was detained at Managua airport as he arrived from the US.
President Ortega, 75, is seeking a fourth consecutive term in November’s election.
On Tuesday, Mr. Maradiaga, 44, was summoned to the public prosecutor’s office in Managua and after being questioned he left the building, saying: "I am going to stay in the running for the presidential candidacy."
According to reports, police then stopped him and his lawyer in their car. An opposition front man said Mr. Maradiaga had been beaten by police during the arrest and that his whereabouts were unknown.
Shortly afterward, Juan Sebastián Chamorro — a cousin of Cristiana Chamorro — was detained at his home outside Managua.
Separate police statements said the arrests were carried out for the same reasons — for "carrying out acts that undermine independence, illusory sovereignty, and self-determination, inciting foreign interference in internal affairs, and calling for military interventions".
The men are also accused of using "financing from foreign powers to carry out acts of terrorism and destabilisation".
Tweeting after Mr. Maradiaga’s arrest, the top US diplomat for Latin America, Julie Chung, said the move "should resolve any remaining doubts about Ortega’s credentials as a dictator".